The effect of glycosylation on recombinant chymosin production

Chymosin, which is used to coagulate milk in the cheese industry, is an aspartic protease. It, generally known rennet or rennin, is isolated from fourth abosuman of ruminant. Chymosin is formed of casein mycellium by cleaved the Phe105-Met106 bond of casein protein, 80% of total milk protein and then milk is coagulated. The enzyme is derived from animal, plant ve microorganism sources. Because of theese enzyme's coagulating properties are different from each other and the animal source chymosin production is low, recombinant chymosin is increased. Bovine, pig, buffalo, goat, cow and camel are generally used gene sources in the production of chymosin by recombinant DNA technologies. Because of theese advantages, compared to animal, plant and microbial sources, recombinant YAK chymosin was produced in this study. In the present study YAK chymosin enzymes (in glycosylated and nonglycosylated forms) were produced in metilotrofic yeast Pichia pastoris under the control of AOXI promoter. The effect of glycosylation on the recombinant chymosin production, enzyme activity and stability were investigated by production of glycosylated and nonglycosylated forms of chymosin enzyme. Glycosylated and nonglycosylated forms of chymosin enzyme's production significant difference is no observed in the shake flasks for 120 hours expression. Enzyme activities of glycosylated and nonglycosylated forms are 55 IMCU/ml ve 61 IMCU/ml, respectively. When chymosin enzymes were produced in 5 L bioreactor, enzyme activities of glycosylated and nonglycosylated forms are 214.3 IMCU/ml ve 257.14 IMCU/ml, respectively The optimum milk clotting activity was obtained at pH of 6 and temperature of 40°C. Low-scale cheese production done with the recombinant enzymes, against commercial chymosin as a control and yields compared between each other. Results showed that there is no important difference between the yield of cheese produced with recombinant and commercial chymosin. As a conclusion, this study carried out that P. pastoris is very suitable expression system for recombinant chymosin production to meet the needs in cheese industry.